Elsevier S & T

Apoptosis

Wilson, Leslie

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Table of contents

Cover
Contentsv
Contributorsxi
Prefacexv
Chapter 1. Isolation and Assay of Caspases1
I. Introduction1
II. Materials Required5
III. Methods6
References25
Chapter 2. Cloning and Analysis of Bcl-2 Family Genes29
I. Introduction29
II. Cloning of Bcl-2 Family Genes33
III. Analysis of Bcl-2 Family Protein Function and Regulation36
References45
Chapter 3. Flow Cytometric Analysis of Cell Shrinkage and Monovalent Ions during Apoptosis49
I. Introduction49
II. Analysis of Cell Size by Flow Cytometry52
III. Analysis of Monovalent Intracellular Ions by Flow Cytometry57
IV. Sorting Apoptotic Cells by Flow Cytometry64
References67
Chapter 4. Use of Flow and Laser-Scanning Cytometry in Analysis of Cell Death69
I. Introduction70
II. Principles of Cell Measurement by Laser-Scanning Cytometry (LSC)72
III. Specifics of Cell Preparation for Analysis by LSC75
IV. Chromatin Condensation as a Marker of Apoptotic Cells Detected by LSC77
V. Gross Changes in Cell Structure during Apoptosis Measured by Laser-Light Scattering79
VI. Dissipation of Mitochondrial Transmembrane Potential (Delta Phi m) during Apoptosis80
VII. Annexin V as a Marker of Apoptotic Cells84
VIII. DNA Fragmentation Assay: Detection of Cells with Fractional (SubdiploidŽ) DNA Content87
IX. DNA Fragmentation: Detection of DNA Strand Breaks (TUNELŽ Assay)90
X. Analysis of Caspase Activation by the Labeled Inhibitor Method93
XI. Detection of Apoptotic Cells Based on Cleavage of Poly(ADP-ribose) Polymerase97
XII. Unique Possibilities Offered by LSC in Studies of Apoptosis100
XIII. Strategies in Analysis of Apoptosis by Flow Cytometry or LSC102
References105
Chapter 5. Analysis of Protein Transglutamylation in Apoptosis111
I. Introduction112
II. Transglutaminase (TGase) Activity Assays114
III. Determination of ε-(y-Glutamyl)-lysine Cross-Link Density in Apoptotic Tissue115
IV. Immunoprecipitation of TGase-Interacting Proteins from Apoptotic Cells119
V. In Situ Identification of TGase Glutamyl Donor Substrate Proteins in Apoptotic Tissue119
VI. Identification of Labeled Substrate Proteins124
VII. Identification of TGase Reactive Glutamine Residues by Protein Sequencing125
VIII. Location of Cross-Links in Protein Complexes126
IX. Analysis of TGase-Mediated Glutamine Deamidation by Peptide Sequencing130
X. Reagents131
References132
Chapter 6. Analysis of Sphingomyelin and Ceramide Levels and the Enzymes Regulating their Metabolism135
I. Introduction136
II. Metabolic Labeling136
III. Determination of Sphingomyelin Mass Levels142
IV. Enzymatic Method for the Quantitation of Ceramide-Diacylglycerol (DAG) Kinase Assay144
V. Chromatographic Quantitation of Ceramide Using HPLC and Fluorescence Spectroscopy147
VI. Assays for Enzymes Regulating Cellular Levels of Sphingomyelin and Ceramide149
References161
Chapter 7. Cell-Free Systems to Study Apoptosis167
I. Introduction167
II. Preparation of Extracts168
III. In Vitro Apoptosis Assays171
IV. Factors That Affect Caspase Activation in Extracts181
V. Concluding Remarks183
References183
Chapter 8. Role of c-Jun N-terminal Kinase in Apoptosis187
I. Introduction187
II. Methods for the Detection of c-Jun N-terminal Kinase ( JNK) Activation during Apoptosis190
III. Evaluation of the Role of JNK Activation in Apoptosis193
References193
Chapter 9. Methods for Studying Pro- and Antiapoptotic Genes in Nonimmortal Cells197
I. Introduction198
II. Mouse Embryo Fibroblasts (MEFs)199
III. Retroviral-Mediated Gene Transfer202
IV. Basic Infection Protocol for MEFs (Using the Phoenix System)205
V. Analysis of Cells210
VI. Other Systems222
References225
Chapter 10. Calcium Flux Measurements in Apoptosis229
I. Background229
II. Ca2+-Specific Probes234
III. Calcium Imaging237
IV. Summary and Conclusions242
References243
Chapter 11. Proteinase Families and Their Inhibitors247
I. Introduction247
II. Enzyme Classification248
III. Proteinase Inhibitors249
IV. Considerations for Inhibitor Selection and Application259
V. General Guidelines for Using Proteinase Inhibitors265
VI. Concluding Remarks266
References267
Chapter 12. Identification and Analysis of Caspase Substrates: Proteolytic Cleavage of Poly(ADP-ribo289
I. Introduction290
II. Poly(ADP-ribose)polymerase (PARP-1)291
III. DNA Fragmentation Factor II (DFF45)300
References303
Chapter 13. Analysis of Reactive Oxygen Species in Cell Death307
I. Introduction307
II. Methods309
References318
Chapter 14. Methods for Studying Apoptosis and Phagocytosis of Apoptotic Cells in Drosophila Tissues321
I. Introduction321
II. Detection of Apoptosis in Drosophila Tissues323
III. Phagocytosis330
IV. Testing for Apoptotic Activities in Drosophila S2 Cells332
References338
Chapter 15. Phosphatidylserine Exposure and Phagocytosis of Apoptotic Cells339
I. Introduction340
II. Annexin V Purification342
III. Annexin Staining for Flow Cytometric Analysis of Apoptotic Cells346
IV. In Vivo Detection of Cell Surface-Exposed PS349
V. MC540 Staining354
VI. Transbilayer Lipid Movement Assays355
VII. Phagocytosis of Apoptotic Cells358
References362
Chapter 16. The (Holey) Study of Mitochrondria in Apoptosis365
I Introduction366
II. Induction of Cytochrome c Release from Mitochrondria in Cultured Cells368
III. Assessment of Cytochrome c Release in Heterogeneous Populations of Cultured Cells369
IV. Assessment of Cytochrome c Release in Single Cells372
V. Assessment of Mitochondrial Cytochrome c Release in Cell-Free Systems375
VI. Detection of Proteins Released from Mitochondria Concomitant with Cytochrome c377
VII. Assessment of Mitochondrial Function379
References386
Appendix390
Chapter 17. In Situ Detection of Dying Cells in Normal and Pathological Tissues393
I. Introduction394
II. Morphological Features of Cell Death: Apoptosis vs Necrosis395
III. Methods for Apoptotic Cell Identification397
IV. TUNEL397
V. ISEL402
VI. Comments for Both TUNEL and ISEL405
VII. Other Methods406
VIII. General Considerations410
References412
Chapter 18. Model Cell Lines for the Study of Apoptosis in Vitro417
I. Introduction418
II. Jurkat424
III. 2B4.11426
IV. DO11.10427
V. MCF-7428
VI. PC12429
VII. C2C12430
References431
Chapter 19. Programmed Cell Death Assays for Plants437
I. Introduction438
II. DNA Fragmentation by Gel Electrophoresis438
III. TUNEL443
IV. Nondestructive Measurements of Plant Cell Senescence447
References450
Chapter 20. Studies of Apoptosis Proteins in Yeast453
I. Introduction453
II. Methods456
III. Inhibition of Caspase- and Bax-Induced Cell Death in Yeast465
IV. Comments and Conclusions466
References467
Chapter 21. Methods to Study Cell Death in Dictyostelium discoideum469
I. Introduction470
II. Cell Culture and Maintenance of Axenic Strains473
III. Induction of Developmental Cell Death476
IV. Analysis of Cell Death Characteristics480
V. Genetic Manipulation of Dictyostelium485
VI. Selection and Analysis of Cell Death-Resistant Mutants491
References494
Chapter 22. Methods of Study of Tumor Necrosis Factor-Related Ligands in Apoptosis499
I. Introduction500
II. Production and Characterization of Reagents503
III. Receptor Binding Characteristics of TNF Family Ligands512
IV. Induction of Apoptosis517
V. Effect of Viral Infection on Cell Susceptibility to TNF Ligand-Mediated Cytotoxicity520
References524
Index527
Volumes in Series547

Book details

Vendor Elsevier S & T
SKU 9780125441650
ISBN-13 9780080496610
Author Wilson, Leslie
Edition 2nd
Category Science
Subject Cell Biology

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Apoptosis provides a current and comprehensive collection of methods for the study of cell death. Using a diverse range of technical approaches and model systems, the chapters in this volume cover topics from the cellular and organismal to the molecular and anatomical. The methods are illustrated with user-friendly recipes and over 100 tables, halftones, and diagrams.

Key Features
* Current methodologies for studying cell death
* Wide range of model systems
* Molecular, biochemical, cellular, and genetic approaches
* Complements the original Cell Death volume
* Up-to-date methodology for a fast moving field
* Designed with the needs of both basic scientists and clinicians in mind
* Authors are leaders in their respective fields

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